Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Endpoint summary

Description of key information

Physical-chemical properties and results of in vivo mammalian toxicity studies indicate that Nigrosine is absorbed via the gastro-intestinal tract . Dermal and inhalation are not the most significant route of exposure for this substance. In the absence of any reliable quantitative data, 100 % oral, dermal and inhalation absorption will be used for risk assessment purposes.

 

The lipophilic nature of the registered substance suggests a preferential distribution to tissues with a high lipid content.

 

Metabolism of absorbed material is expected to produce mainly nitrophenols and aminophenols, which will then be conjugated with glucuronide and/or sulphate, and ring-cleavage products such as 4-nitrophenylmercapturic acid, and then easily excreted in the urine. Any test material that is not absorbed will be excreted in the faeces, as evidenced in the repeated dose toxicity studies.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

100

Absorption rate - inhalation (%):

100

Additional information

INTRODUCTION

In accordance with the section 8.8.1 of Annex VIII in Regulation (EC) No 1272/2008, the toxicokinetic profile of Nigrosine (reaction products of benzamine, aniline hydrochloride and nitrobenzene) was derived from all available information collated in the dossier. In the absence of experimental studies of the absorption, distribution, metabolism or elimination, the physicochemical properties of the substance and the results of mammalian toxicity studies were used to assess the toxicokinetic properties of the substance.

 

PHYSICOCHEMICAL PROPERTIES

The substance Nigrosine is a complex reaction product, and is defined under REACH as a substance of Unknown or Variable Composition, Complex reaction product (UVCB). It is a black powdery solid, with 15.6 % having an inhalable particle size of less than 100 µm (sieve method), 0.741 % a thoracic particle size of less than 10.0 µm (cascade impactor method), and 0.05 % a respirable particle size of less than 5.5 µm (cascade impactor method). It is sparingly water soluble (less than 0.1 mg/L at 20.0 ± 0.5 °C) with a log Pow range of 0.518 to 6.50 (by percentage area normalisation, 96.2 % of the test material components had a log Pow greater than 3.00 and 39.2 % a log Pow greater than 6.50).

 

Estimations of the dissociation constants using the ACD/pKa 8.03 indicated that the benzene substituted cyclic amine functional groups have dissociation constants of 6.19 and 6.57 respectively, thus accepting protons at an acidic pH. When the additional R groups were aniline molecules, the resulting secondary amines functional groups were estimated to accept additional protons only under extremely acidic conditions, with dissociation constant estimates of -1.41 and -2.52 respectively.

 

ABSORPTION:

Oral absorption

Given the multiple component nature of the substance, different components will be absorbed to a different extent, however, the overall very poor water solubility and rather high lipophilicity, the large particle size, and the possibility of ionisation in acidic conditions suggest minimal oral absorption. This is supported by the results of two acute oral toxicity studies, in which no signs of systemic toxicity were observed after dosing with either 5 000 or 2 000 mg/kg, and no staining of systemic tissues was observable (the material is used as a pigment).

The results of the repeated 28-d toxicity study, the 90-day repeated dose toxicity study as well as those of the reproduction/developmental toxicity screening test (OECD 421) and of the pre-natal developmental toxicity studies in rats confirm minimal absorption following repeated dosing. Both in the 28-d toxicity study and 90-day study, the treatment at the dose level of 1000 mg/kg/day resulted in haematological effects and associated splenic histopathological changes in rats, likely related to absorption of minimal amounts of the aniline contained in the substance. A sign of absorption was also apparent with the liver weight and histopathological findings in the 28-day repeated dose toxicity study. Based on this information, oral absorption of the substance is likely, but the exact extent cannot be determined. Therefore, in accordance with the ECHA guidance, the oral absorption rate is assumed to be 100% for the purpose of human health risk assessment.

 

Dermal absorption

Physical-chemical properties of the registered substance all infer minimal dermal absorption; the lack of any clinical signs in the acute dermal toxicity study and the absence of skin sensitising properties support this assumption. However, the dermal absorption of 100% is assumed for the substance in the absence of specific information.

 

Inhalation absorption

Clinical signs, such as the increased respiratory rate, hunched posture and pilo-erection, were observed following the 4-hour exposure to a dust atmosphere of the substance to the rats with macropsopic abnormalities apparent in the lungs, which would indicate the systemic absorption of substance. In the absence of any data on absorption of inhaled nigrosine, inhalation absorption is set at 100 %.

 

DISTRIBUTION 

The lipophilic nature of the registered substance suggests a preferential distribution to tissues with a high lipid content. Following oral administration, black contents, associated with the colour of the material, were seen in the gastro-intestinal tract of rats as well as in the faeces. No such blackish colour was reported in any other organs that would otherwise have indicated the distribution of the substance.

 

METABOLISM

Components within the registered substance will be mainly subjected to ring oxidation, producing products as nitrophenols and aminophenols, which will be then conjugated with glucuronide and/or sulphate, and ring-cleavage products such as 4-nitrophenylmercapturic acid. Reduction to produce free aniline cannot be excluded, but the results of the 28-day repeated dose toxicity study, the 90-day repeated dose toxicity study, and of the OECD 421 and pre-natal developmental toxicity studies in rats showed that this event would be minimal, because methaemoglobin production was very limited in the 28-d toxicity study, even at the limit dose of 1 000 mg/kg/day. The majority of the mean and individual haematological parameters that showed statistically significant changes were within the range of the laboratories historical control data or only slightly outside the ranges. Neither the mothers nor the foetuses showed any sign of hypoxic suffering at 1000 mg/kg/day in the pre-natal developmental study. Finally, mutagenicity studies confirm neither the UVCB nor potential rat metabolites of the UVCB components confirm a lack of direct DNA reactivity, as the results for all in vitroend points were clearly negative in both the absence and presence of metabolic activation.

 

ELIMINATION 

Urine is expected to represent the major route of elimination from the body for the majority of conjugate products. Since no notable kidney weight changes were observed in the available repeated dose toxicity studies in rats; biliary excretion may well be another route of excretion for this low water-soluble substance. Any test material that is not absorbed will be excreted in the faeces, as evidenced with the blackish faeces in the repeated dose toxicity studies in rats. Given the high Log Pow range of the substance, it is expected that any substance, which has penetrated the stratum corneum but not penetrated the viable epidermis may be sloughed off with skin cells.

 

CONCLUSION 

Physical-chemical properties and results of in vivomammalian toxicity studies indicate that Nigrosine is absorbed via the gastro-intestinal tract . Dermal and inhalation are not the most significant route of exposure for this substance. In the absence of any reliable quantitative data, 100 % oral, dermal and inhalation absorption will be used for risk assessment purposes.

 

The lipophilic nature of the registered substance suggests a preferential distribution to tissues with a high lipid content.

 

Metabolism of absorbed material is expected to produce mainly nitrophenols and aminophenols, which will then be conjugated with glucuronide and/or sulphate, and ring-cleavage products such as 4-nitrophenylmercapturic acid, and then easily excreted in the urine. Any test material that is not absorbed will be excreted in the faeces, as evidenced in the repeated dose toxicity studies.

REFERENCES

Allen D.J. (1993a)

Nigrosine Base EEL: Acute oral toxicity (limit test) in the rat

Safepharm Laboratories Ltd. project No. 370/68

GLP

Unpublished

 

Allen D.J. (1993b)

Nigrosine Base EEL: Acute dermal irritation test in the rabbit

Safepharm Laboratories Ltd. project No. 370/70

GLP

Unpublished

 

Allen D.J. (1993c)

Nigrosine Base EEL: Magnusson & Kligman maximisation study in the guinea pig

Safepharm Laboratories Ltd. project No. 370/72

GLP

Unpublished

 

Griffiths D.R. (2007)

Nigrosine Base SALP: Acute inhalation toxicity (nose only) study in the rat

Safepharm Laboratories Ltd. project No. 0370/0353

GLP

Unpublished

 

Namiki M. (2007)

Preliminary Reproduction Toxicity Screening Study of NIGROSINE BASE SALP by Oral Administration in Rats

Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd. study No. B060725

GLP

Unpublished

 

O’Connor B.J. and Mullee D.M. (2007)

Nigrosine Base SALP: Determination of general physico-chemical properties

Safepharm Laboratories Ltd. project No. 0370/0351

GLP

Unpublished

 

Oroszlány, 2019

Nubian black TN-870: a 90-day oral (gavage) toxicity study in Wistar rats

CitoxLAB Study No. 18/076-101P

GLP

Unpublished

 

Van Tuyl Ir. M.H.M. (2007)

A prenatal developmental toxicity study of C.I. Solvent Black 7 in rats by oral gavage

Notox B.V. project No. 469687

GLP

Unpublished

 

Walker D.J. (1992a)

Nigrosine Base: Acute oral toxicity (limit test) in the rat

Safepharm Laboratories Ltd. project No. 370/17

GLP

Unpublished

 

Walker D.J. (1992b)

Nigrosine Base: Acute dermal toxicity (limit test) in the rat

Safepharm Laboratories Ltd. project No. 370/18

GLP

Unpublished

 

Walker D.J. (1992c)

Nigrosine Base EX: Acute dermal toxicity (limit test) in the rat

Safepharm Laboratories Ltd. project No. 370/44

GLP

Unpublished

 

Walker D.J. (1992d)

Nigrosine Base: Acute dermal irritation test in the rabbit

Safepharm Laboratories Ltd. project No. 370/19

GLP

Unpublished

 

Walker D.J. (1992e)

Nigrosine Base EX: Magnusson & Kligman maximisation study in the guinea pig

Safepharm Laboratories Ltd. project No. 370/47

GLP

Unpublished

 

Walker D.J. (1992f)

Nigrosine Base: Magnusson & Kligman maximisation study in the guinea pig

Safepharm Laboratories Ltd. project No. 370/21

GLP

Unpublished

 

Wragg M.S. and Brooks P.N. (1993)

Nigrosine Base EX: twenty-eight day sub-acute oral (gavage) toxicity study in the rat

Safepharm Laboratories Ltd. project No. 370/133

GLP

Unpublished